EP0506377B1 - Calmodulin bindendes Protein - Google Patents

Calmodulin bindendes Protein Download PDF

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EP0506377B1
EP0506377B1 EP19920302591 EP92302591A EP0506377B1 EP 0506377 B1 EP0506377 B1 EP 0506377B1 EP 19920302591 EP19920302591 EP 19920302591 EP 92302591 A EP92302591 A EP 92302591A EP 0506377 B1 EP0506377 B1 EP 0506377B1
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sequence
caldesmon
dna
polypeptide
cells
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EP0506377A3 (en
EP0506377A2 (de
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Ken'ichiro Hayashi
Kiyozo Asada
Takashi Hashida
Hirokazu Kotani
Ikunoshin Kato
Kenji Sobue
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Takara Shuzo Co Ltd
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Takara Shuzo Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • This invention relates to a polypeptide that has the amino acid sequence of the functional domain of human caldesmon.
  • Caldesmon is a protein that can bind with calmodulin, actin and tropomyosin, found in all tissues except skeletal muscle and cardiac muscle; it contributes to the regulation of actomyosin system in smooth muscle. This regulatory function depends on the concentration of calcium ions (flip-flop regulation) in bringing about the regulation of the actomyosin system (Proc. Natl. Acad. Sci. USA, 78 , 5652-5655, 1981). When the concentration of calcium ions is low, caldesmon binds with the actin filament-tropomyosin system, and inhibits the mutual interactions of actin and myosin. When the calcium ion concentration is increased, complexes of active calmodulin and caldesmon are formed. This complex is released from the actin filament-tropomyosin system. Because of this, the inhibition of actin-myosin interaction caused by caldesmon cease, and these interactions begin.
  • Caldesmon was first isolated from the smooth muscles of chicken gizzards, and it has been isolated from other vertebrates. The results of the limited digestion of chicken caldesmons by the protease ⁇ -chymotrypsin have shown that a polypeptide of the size of about 35 kDa at the carboxy-terminal end of chicken caldesmons is the functional unit that brings about flip-flop regulation that depends on the concentration of calcium ions (J. Biochem., 102 , 1065-1073, 1987).
  • Human platelet caldesmon 77 has been purified and used to prepare antibodies to use in the domain mapping of chicken gizzard caldesmon, Journal of Biological Chemistry, 262 , No. 6, 25 February 1987, 2757-2763.
  • cDNA clones spanning a coding region of 461 amino acids of low M ⁇ human caldesmon have been obtained, J. CELL BIOL. 111 (5 Part 2), 163A, Abstract No 894, 1990.
  • a cDNA probe encoding the C-terminus of avian caldesmon has been used to screen a human aorta library and clone smooth-muscle and non-muscle caldesmon-encoding cDNAs. The predictions from this are that the smooth-muscle protein is 793 aa long and the non-muscle protein, missing the central helical domain of 256 aa, is 537 aa long, Gene 112 (1992) 197-204.
  • the morphological change of cells is one characteristic of carcinogenesis.
  • the preservation of cell morphology is closely related to the actin network of the cells, and when cells are transformed, the change in cell morphology occurs in correlation with loss of actin cables, which are part of the filament system that makes up the cytoskeleton (Proc. Natl. Acad. Sci. USA, 72 , 994-998, 1975).
  • Immunohistochemical analysis have revealed that caldesmon is localized in actin cables, cell attachment sites, and membrane ruffles in normal cells, but in cancer cells, the compound is not found in any defined cell location (Proc. Natl. Acad. Sci. 81 , 3133-3137, 1984).
  • this polypeptide could be used to inhibit or accelerate the mutual interactions between actin and myosin whatever the calcium concentration is, so that the polypeptide could be used as a vasodilator or a regulator of the movements of the digestive tract.
  • the purpose of this invention is to provide a polypeptide that has the activity of human caldesmon and the DNA sequence that codes for said polypeptide.
  • the first part of this invention relates to a polypeptide that has a calmodulin-binding activity and an actin-binding activity characterized by said polypeptide has an amino acid sequence of sequence ID No.1 in the sequence listing in a polypeptide molecule.
  • the second part of this invention relates to a gene that codes for the polypeptide of the first part of this invention.
  • the polypeptide of this invention is a polypeptide that has both calmodulin-binding activity and actin-binding activity; it is, for example, a polypeptide that has within it the amino acid sequence shown as sequence ID No.1 in the sequence listing, which sequence is that of the functional unit of human caldesmon, which unit was discovered by the inventors of this invention.
  • Polypeptides that have within them this amino acid sequence include, for example, the polypeptides that have the sequence ID No.2 to 5 of the sequence listing.
  • a gene that codes for the polypeptide of artificial human caldesmon is, for example, a gene with the DNA sequence of sequence ID No.7 to 12 in the sequence listing, and genes that can hybridize to the gene with the sequence ID No.7 to 12, and which genes code for a polypeptide that has calmodulin-binding activity and actin-binding activity.
  • the inventors of this invention prepared a cDNA library from HeLa cells, a human cell line, and next selected cDNA clones that code for a polypeptide of human caldesmon from this cDNA library; the DNA sequence and the amino acid sequence coded for human caldesmon were deduced by DNA sequence analysis. Next, based on various finding, the inventors prepared polypeptides of various lengths, and looked for the functional domain of human caldesmon polypeptide, using calmodulin-binding activity and actin-binding activity as indices. Based on the findings described below, the inventors achieved this invention.
  • the gene that codes for human caldesmon can be isolated and analyzed as described here.
  • HeLa cells or the like can be used, and the total RNA, which includes the poly(A) + RNA, of the cells, was isolated, and bound onto a cellulose carrier or the like with oligo(dT). This was used as a template in the synthesis of cDNA with reverse transcriptase.
  • the cDNA was synthesized by the method of Okayama-Berg or by the method of Gubler-Hofmann.
  • the cDNA synthesized in this was ligated with a plasmid or phage vector and introduced into a host, giving a cDNA library.
  • the cDNA library was screened for the desired clones by one of the two following methods.
  • the amino acid sequence of a portion of the desired protein was identified, and an oligonucleotide that coded for that amino acid sequence was synthesized and used as a probe in screening.
  • an antibody that bound with the desired protein was prepared and used to screen the cDNA library constructed in an expression vector, in an immunological method.
  • ⁇ gtl1 When the immunological method of screening is used, ⁇ gtl1 is well used as the expression vector.
  • ⁇ gt11 is commercially available; it can be purchased from, for example, Stratagene.
  • IPTG isopropyl- ⁇ -D-galactopyranoside
  • the filter is immersed in buffer that contains bovine serum albumin (below, referred to as the blocking buffer), and after this step of blocking, antibodies (first antibody) that bind with the desired protein are added to fresh blocking buffer, and the filter is immersed in this blocking buffer, so that the desired protein and the antibodies form complexes.
  • the filter is immersed in a blocking buffer that contains the second antibody, labelled with an enzyme.
  • the first antibody and the second antibody form complexes.
  • Second antibodies coupled to alkaline phosphatase or peroxidase are commercially available.
  • the filter is washed to remove excess second antibody that has not adsorbed to the filter, and the filter is put in a developing solution, and colored clones are selected.
  • the recombinant phages obtained in this way are used to infect host Escherichia coli cells, and phage DNA is obtained from the phage lysate.
  • the cDNA from the recombinant phage DNA is isolated and purified, and its DNA sequence is identified.
  • the antibodies used for immunological screening can be antisera prepared in rabbits immunized with other kinds of caldesmon, such as chicken caldesmon that have amino acid sequences likely to correspond in part to the sequence of the desired protein, human caldesmon.
  • the cDNA sequence coding for human caldesmon which has been identified by the inventors, is sequence ID No.13 and 14 in the sequence listing.
  • sequence ID No.13 and 14 The cDNA sequence coding for human caldesmon, which has been identified by the inventors, is sequence ID No.13 and 14 in the sequence listing.
  • the 558 amino acid residues shown as sequence ID No.6 in the sequence listing is that of the caldesmon of high molecular weight
  • the DNA sequence that codes for this is sequence ID No.12 of the sequence listing.
  • the caldesmon of low molecular weight is exactly the same as that of high molecular weight except that it lacks amino acids 202 to 227, and it is shown as the 532 amino acids of sequence ID No.5 in the sequence listing, and the DNA sequence that codes for this is sequence ID No.11 in the sequence listing.
  • the form of higher molecular weight is called type I
  • the form of lower molecular weight is called type II.
  • DNA that codes for the caldesmon of type I and DNA that codes for the caldesmon of type II are prepared separetely from the recombinant phage DNA mentioned above, and the DNA is inserted into an expression plasmid, such as the plasmid pTV118N.
  • the plasmid that coded for the caldesmon of type I was designated pTV118NHS3CaDl
  • the plasmid that coded for the caldesmon of type II was designated pTV118NHS3CaD2.
  • pTV118NHS3CaDl it is possible to prepare DNA that codes for polypeptides of various lengths.
  • a site slightly upstream of the initiation codon of the region that codes for type I caldesmon of this plasmid can be cleaved with an appropriate restriction enzyme, and exonuclease can be used to remove the sequence of the 5'-side in the caldesmon gene.
  • exonuclease can be used to remove the sequence of the 5'-side in the caldesmon gene.
  • PCR can be done (at 94°C for 30 sec in step 1, at 55°C for 2 min in step 2, and at 72°C for 1 min in step 3, for a total of 20 cycles), which results in the amplification of DNA that contains the DNA sequence shown as sequence ID No.10 in the sequence listing, which codes for the polypeptide shown in the sequence listing as having sequence ID No.4 (called 312AA below).
  • sequence ID No.17 and 18 in the sequence listing as primers it is possible to amplify DNA that contains the DNA sequence shown as sequence ID No.9 in the sequence listing, which codes for the polypeptide shown in the sequence listing as having sequence ID No.3 (called 122AA below).
  • sequences ID No.18 and 19 in the sequence listing as primers it is possible to amplify DNA that contains the DNA sequence shown as sequence ID No.8 in the sequence listing, which codes for the polypeptide shown in the sequence listing as having sequence ID No.2 (called 118AA below).
  • sequence ID No.20 and 21 in the sequence listing as primer it is possible to amplify DNA that contains the DNA sequence shown as sequence ID No.23 in the sequence list, which codes for the polypeptide shown in the sequence listing as having sequence ID No.22 (called 94AA below).
  • sequences ID No.21 and 24 in the sequence listing as primers, it is possible to amplify DNA that contains the DNA sequence shown as sequence ID No.26 in the sequence listing, which codes for the polypeptide shown in the sequence listing as sequence ID No.25 (called 90AA below). Next, these DNAs are inserted into expression plasmids, such as, for example, pTV118N.
  • the plasmid that coded for 312AA was named pTHCD247
  • the plasmid that coded for 122AA was named pTHCDXa443
  • the plasmid that coded for 118AA was named pTHCD443
  • the plasmid that coded for 94AA was named pTHCDXa451
  • the plasmid that coded for 90AA was named pTHCD451.
  • the plasmids were used to transform Escherichia coli cells, such as E . coli JM109, for their expression, and the recombinant cells were cultured under appropriate conditions so that the desired polypeptides accumulated within the E . coli cells.
  • the purification of the polypeptides was, for example, as follows.
  • the recombinant E . coli cells were cultured in a culture medium such as L broth, and the cells were harvested and disrupted by being sonicated. The disrupted cells were centrifuged and the supernatant was obtained. After treatment such as dialysis, the dialysate was put on a column for gel filtration and then on a column for ion-exchange chromatography for purification.
  • the desired proteins were purified by use of affinity columns coupled with calmodulin or tropomyosin, making use of the properties of the target protein.
  • 122AA and 94AA have a sequence that can be recognized by factor Xa. By cleaving with factor Xa site-specifically, it is possible to prepare a polypeptide shown as sequence ID No.1 in the sequence listing (called 116AA below) and a polypeptide shown as sequence ID No.27 in the sequence listing (called 88AA below).
  • Type II is a polypeptide that is missing one portion of type I.
  • 312AA is a polypeptide that is the C-terminal portion of types I and II
  • 122AA and 118A are polypeptides that have a sequence of 116 amino acids in the C-terminal sequence of 312AA
  • 94AA and 90AA are polypeptides that have a sequence of 88 amino acids that are in the central portion of 118AA, lacking both the N-terminal portion and the C-terminal portion.
  • the polypeptide (typeI, typeII,312AA, 122AA, 118AA, and 116AA) that contains the polypeptide of 116 amino acids (called 116AA below) that has sequence ID No.1 in the sequence listing has both calmodulin-binding activity and actin-binding activity.
  • Type I, type II, 312AA, 122AA, 118AA, 116AA all have tropomyosin-binding activity and have a inhibitory activity to actomyosin ATPase, and so the functional unit of human caldesmon is identified as being 116AA.
  • this invention provides various polypeptides that have activities of human caldesmon and also provides genes that code for these polypeptides. It is possible to produce both on a large scale by the use of genetic engineering, and said genes and polypeptides will be useful in diagnosis and other fields of medicine, and in biochemical research, as well.
  • the functional unit of human caldesmon, 116AA can be used as a material for chimera proteins and peptide transfer, and the DNA with sequence ID No.7 in the sequence listing will be of use in the fields of genetic engineering and protein engineering.
  • RNA of HeLa cells was obtained by the method of guanidium-cesium chloride (Biochemistry, 18 , 5294-5299, 1979), and poly(A) + RNA was isolated by column chromatography with oligo-(dT)cellulose.
  • the purified poly(A) + RNA was used as a template and oligo-(dT) was used as the primer in the synthesis of cDNA by the method of Gubler-Hoffmann (Gene, 25 , 263-269, 1983).
  • the ends of the cDNA were blunted with T4 DNA polymerase, and the Eco RI site in the cDNA was methylated with Eco RI methylase.
  • cDNA was ligated to an Eco RI linker [d(pGGAATTCC)] with use of T4 DNA ligase, and cDNA with both ends having an Eco RI was constructed by Eco RI digestion.
  • This cDNA was ligated with the Eco RI arm of ⁇ gt11 (Stratagene), and a cDNA library was made by in vitro packaging with GigapackII Gold (Stratagene).
  • Plaques of phages with use of E . coli Y1090 as the host cells were formed. To do this Y1090 cells were cultured overnight at 37°C in L medium that contained 0.02% maltose, and the cells were collected by centrifugation. The cells harvested were suspended in 10 mM MgSO 4 and were added into a suspension of phage solution and kept at 37°C for 15 minutes to allow attachment of the phages to the host cells.
  • the nylon membrane was removed from the plate and immnologically screened with rabbit anti-caldesmon antibodies obtained by the immunization of a rabbit with 35kDa fragment of chicken caldesmon obtained by the digestion, with ⁇ -chymotrypsin (J. Biochem., 102 , 1065-1073, 1987) and with F(ab') 2 fragment of goat anti-rabbit immunoglobulin antibody labelled with alkaline phosphatase as the second antibody. Clones that were stained with nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphate (BCIP) were positive. Several positive signals were obtained from among the 250,000 clones.
  • NBT nitro blue tetrazolium
  • BCIP 5-bromo-4-chloro-3-indolylphosphate
  • plaques that corresponded to the positive signals were cut out together with agar and suspended in 500 ⁇ l of SM solution (50 mM Tris-HCl, 100 mM NaCl, and 10 mM MgSO 4 , pH 7.5), plated after being diluted appropriately, and screened again as described above, by which procedure it was possible to isolate four phage clones independently.
  • SM solution 50 mM Tris-HCl, 100 mM NaCl, and 10 mM MgSO 4 , pH 7.5
  • E . coli Y1090 cells that had been cultured overnight in L medium were harvested and suspended in 10 mM MgSO 4 .
  • the cell suspension was mixed with the phage solution mentioned above at multiplicity of infection (moi) of 0.01, and the mixture were left at 37°C for 15 minutes.
  • the mixture of phages and E . coli cells was used to inoculate L medium that contained 10 mM MgSO 4 , and continued to be cultured at 37°C until a considerable amount of bacterial debris remained after lysis.
  • sodium chloride was added to the final concentration of 0.5 M, chloroform was added to the final concentration of 0.5%, and the mixture was stirred at 37°C for 15 minutes.
  • the supernatant obtained by centrifugation was mixed with polyethylene glycol 6000 added to the final concentration of 10% (w/v), and the mixture was left overnight at 4°C.
  • the phage precipitate obtained by centrifugation was dissolved in TM solution (50 mM Tris-HCl and 10 mM MgSO 4 , pH 7.8), and by the method of glycerol step-gradient ultracentrifugation (T.Maniastis et al., Molecular cloning: A laboratory manual, pp. 83-84, Cold Spring Harbor Laboratory, 1982), the phages were purified.
  • the phages obtained were suspended in TM solution, and DNase I and RNaseA were added to the suspension, which was then kept at 37°C for 30 minutes before the addition of EDTA, Proteinase K (Sigma), and SDS to the concentrations of 20 mM 50 ⁇ g/ml, and 0.5%, respectively. After then the mixture was kept at 65°C for 1 hour. Phenol extraction was done, followed by extraction with diethyl ether, and to the aqueous layer, a 1/10 volume of 5 M sodium chloride and two volumes of ice-cold ethanol were added, which caused precipitation of the DNA (below, this step is referred to as ethanol precipitation). The mixture was centrifuged and the precipitated DNA was collected, after which it was washed in 70% ethanol and dried before being dissolved in 100 ⁇ l of TE solution (10 mM Tris-HCl and 1 mM EDTA, pH 8.0).
  • the recombinant ⁇ gt11 DNA prepared as described above was digested with Eco RI, and the inserted fragments were isolated and purified. Then they were cloned at the Eco RI site of M13mp18RF DNA.
  • the recombinant M13mp18RF DNA was digested with Sal I and Sph I, and by use of exonuclease III (Course in Biochemical Experimentation 1, Methods in genetic research I, pp. 186-200, 1986), a mutant was constructed that lacked various length of sequence at the 5'-side of the Sal I end. Another deletion mutant was prepared that was of recombinant M13mp18RF DNA that had the fragment insertion in the opposite orientation. Then E.
  • coli JM109 cells were transformed with each mutant DNA derived from the recombinant M13mp18 and single-stranded DNA was isolated from the M13mp18 derivatives. Its DNA sequence was identified by the dideoxy-mediated chain-termination method, and the amino acid sequence was deduced from the DNA sequence.
  • Fig. 1 Restriction maps of these clones are shown in Fig. 1.
  • the ATG and TGA shown in the Fig. 1 are the initiation codon and the termination codon for translation, respectively.
  • the caldesmon of higher molecular weight was composed of the 558 amino acid residues shown as sequence ID No.6 in the sequence listing, and the caldesmon of lower molecular weight was composed of the 532 amino acid residues shown as sequence ID No.5 in the sequence listing and was exactly the same as that of higher molecular weight except that it lacked amino acid residues 202 to 227 of the larger caldesmon.
  • the caldesmon of higher molecular weight was named type I
  • the caldesmon of lower molecular weight was named type II.
  • the DNA sequence of type I is that shown as sequence ID No.12 in the sequence listing
  • the DNA sequence of type II is that shown as sequence ID No.11. Their cDNA sequences are shown as sequence ID No.14 and 13, respectively, in the sequence listing.
  • a cDNA fragment of about 800 bp that coded for the N-terminal region of type I caldesmon was obtained by Eco RI digestion of ⁇ gt11HS3CaDN1, and cloned at the Eco RI site of M13mp18RF DNA, giving M13mp18HS3CaDN1.
  • This M13mp18HS3CaDN1 was digested with Nsp (7524)I, blunt-ended with T4 DNA polymerase, and digested with Eco RI, giving cDNA fragments of about 790 bp.
  • This cDNA fragment was cloned in plasmid pTV118N which was digested with Nco I, treated with Klenow fragment (large fragment of E . coli DNA polymerase I), and digested with Eco RI, giving pTV118NHS3CaDN1.
  • cDNA fragments about 1.3 kbp long that coded for the C-terminal region of caldesmon were obtained by Eco RI digestion of ⁇ gt11HS3CaD25, and cloned at the Eco RI site of M13mp18RF DNA, giving M13mp18HS3CaDC. Then M13mp18HS3CaDC was digested with Eco RI and Sac I, giving fragments of cDNA about 940 bp long, which were into pTV118NHS3CaDN1 between the Eco RI and Sac I sites , giving plasmid pTV118NHS3CaD1, which coded for type I caldesmon.
  • E . coli JM109 into which pTV118NHS3CaD1 had been introduced were designated E . coli JM109/pTV118NHS3CaD1, and deposited at the Fermentation Research Institute of the Agency of Industrial Science and Technology, Japan, under FERM BP-3673.
  • T4 DNA polymerase was used to make blunt ends on PCR product A-2, and the product was cloned at the Sma I site of M13mp18RF DNA so as to be in the same direction in the PCR product A-2 as in the M13mp18 lac Z gene. Then cloned M13mp18PCRA-2 with the plasmid A-2 as an insertion was constructed.
  • M13mpl8PCRA-2 was digested with Nsp (7524)I, and after its ends were made blunt with T4 DNA polymerase, it was digested at the multicloning site that originated from M13mp18 with Xba I, and cDNA fragments about 1.6 kbp long were obtained.
  • E . coli cells into which pTV118NHS3CaD2 had been introduced were designated E . coli JM109/pTV118NHS3CaD2, and deposited at the Fermentation Research Institute of the Agency of Industrial Science and Technology, Japan, under FERM P-12013.
  • a primer with sequence ID No.15 of the sequence listing and with the Nco I recognition sequence at its 5'-end and a primer with sequence ID No.16 and with the Sac I recognition sequence at its 5'-end were synthesized with a DNA synthesizer and purified. Said primers were used together with pTV118NHS3CaDl as the template in the PCR to amplify DNA that had the DNA sequence shown as sequence ID No.10 in the sequence listing within its sequence.
  • this amplification product was digested with Nco I and Sac I and extracted with buffered phenol, so that its enzyme activity was lost.
  • the DNA was caused to precipitate with ammonium sulfate and isopropyl alcohol.
  • the precipitated DNA was dissolved with TE solution (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA), and ligated with pTV118N that had been digested with both Nco I and Sac I, giving the plasmid pTHCD247, which codes for 312AA.
  • E . coli JM109 cells into which pTHCD247 had been introduced were designated E . coli JM109/pTHCD247, and deposited at the Fermentation Research Institute of the Agency of Industrial Science and Technology, Japan, under FERM BP-3671.
  • a primer with sequence ID No.19 of the sequence listing and with the Nco I recognition sequence at its 5'-end and a primer with sequence ID No.18 and with the Eco RI recognition sequence at its 5'-end were synthesized with a DNA synthesizer and purified. Said primers were used together with pTV118NHS3CaD1 as the template in the PCR to amplify DNA that had the DNA sequence shown as sequence ID No.8 in the sequence listing within its sequence.
  • this amplification product was digested with Nco I and Eco RI, and treated as in section 3-1 above before being ligated with pTV118N that had been digested with both Nco I and Eco RI, giving the plasmid pTHCD443, which codes for 118AA.
  • E . coli JM109 cells into which pTHCD443 had been introduced were designated E . coli JM109/pTHCD443, and deposited at the Fermentation Research Institute of the Agency of Industrial Science and Technology, Japan, under FERM BP-3672.
  • a primer with sequence ID No.17 of the sequence listing and with the Nco I recognition sequence and the factor Xa recognition sequence at its 5'-end was synthesized with a DNA synthesizer and purified.
  • said primer and the primer described above as having sequence ID No.18 were used together with pTV118NHS3CaD1 as the template in the PCR to amplify DNA that had the DNA sequence shown as sequence ID No.9 in the sequence listing within its sequence.
  • this amplification product was digested with Nco I and Eco RI, and ligated with pTV118N, which had also been digested both Nco I and Eco RI, giving a plasmid that coded for 122AA.
  • Said plasmid was designated pTHCDXa443, and cells of E . coli JM109 into which the plasmid was introduced were named E . coli JM109/pTHCDXa443.
  • a primer with sequence ID No.24 of the sequence listing and with the Nco I recognition sequence at its 5'-end and a primer with sequence ID No.21 and the Eco RI recognition sequence at its 5'-end were synthesized with a DNA synthesizer and purified. Said primers were used together with pTV118NHS3CaD1 as the template in the PCR to amplify DNA that had the DNA sequence shown as sequence ID No.26 in the sequence listing within it.
  • this amplification product was digested with Nco I and Eco RI, and ligated with pTV118N that had been digested with Nco I and Eco RI, giving the plasmid pTHCD451, which codes for 90AA.
  • E . coli JM109 cells into which pTHCD451 had been introduced were designated E. coli JM109/pTHCD451.
  • a primer with sequence ID No.20 of the sequence listing and with the Nco I recognition sequence and the factor Xa recognition sequence at its 5'-end was synthesized with a DNA synthesizer and purified. Said primer and the primer described above as having sequence ID No.21 were used together with pTV118NHS3CaD1 as the template in the PCR to amplify DNA that had the DNA sequence shown as sequence ID No.23 in the sequence listing within its sequence.
  • this amplification product was digested with Nco I and Eco RI, and ligated with pTV118N, which had also been digested both Nco I and Eco RI, giving a plasmid that coded for 94AA.
  • Said plasmid was designated pTHCDXa451, and cells of E . coli JM109 into which the plasmid was introduced were named E . coli JM109/pTHCDXa451.
  • the cells were suspended in E solution (0.3 M KCl, 0.5 mM EGTA, 0.5 mM MgC1 2 , 0.5 mM dithiothreitol (DTT), 0.3 mM PMSF, and 50 mM Tris-HCl, pH 7.0), and the suspension was sonicated to disrupt the cells.
  • the suspension was centrifuged and the supernatant was incubated at 95°C for 5 minutes and then cooled. This was centrifuged and the precipitate was removed.
  • the supernatant was dialyzed against C1 solution (120 mM NaCl, 0.1 mM EGTA, 0.2 mM DTT, and 10 mM Tris-HCl, pH 7.3).
  • the inner solution during dialysis was passed through a column of DEAE Toyopearl 650M equilibrated with C1 solution.
  • calcium chloride was added to the final concentration of 5 mM, the mixture was passed through a column of Sepharose 4B (Pharmacia LKB) coupled with calmodulin which equilibrated with C2 solution (70 mM NaCl, 0.2 mM CaCl 2 , 0.1 mM DTT, and 10 mM Tris-HCl, pH 7.5) as described elsewhere (Journal of Biochemistry, 102 , 1065-1073, 1987), and type I purified was extracted with C2 solution that contained 1.2 mM EGTA and purified.
  • E . coli JM109/pTHCD247 (FERM BP-3671), of E . coli JM109/pTHCDXa443, of E . coli JM109/pTHCD443 (FERM BP-3672), of E . coli JM109/pTHCDXa451 and of E . coli JM109/pTHCD451 were cultured under the same conditions as in the example of 4-1 above, and purification gave 312AA, 122AA, 118AA, 94AA, and 90AA.
  • 122AA and 94AA were treated with restriction proteinase factor Xa (Takara Shuzo Co., Ltd.) for 5 hours at 37°C, and a column of calmodulin-Sepharose was used to purify 116AA and 88AA from the digest.
  • polypeptides purified as described above were mixed with 10 volumes of CaB buffer (10 mM Tris-HCl, pH 7.5, 100 mM KCl, 0.1 mM DTT, and 0.2 mM CaC1 2 ), and put on a column of Sepharose 4B (Pharmacia) coupled with calmodulin which equilibrated with the same buffer. This allowed binding of the polypeptide with calmodulin. After the column was washed with a large amount of the same buffer, the bound polypeptide was eluted from the column with the same buffer, except that 1 mM EGTA was added to the buffer instead of CaC1 2 .
  • CaB buffer 10 mM Tris-HCl, pH 7.5, 100 mM KCl, 0.1 mM DTT, and 0.2 mM CaC1 2
  • the mixture was centrifuged at 200,000 x g for 30 minutes.
  • the monomeric actin remained in the supernatant, whereas the filamentous actin and proteins bound with it (the polypeptide of this invention) precipitated together.
  • the supernatant and the precipitate were separately electrophoresed on SDS-polyacrylamide gels, and the binding of the polypeptide with actin was confirmed.
  • an affinity column with tropomyosin was prepared by the coupling of tropomyosin with Sepharose 4B (Pharmacia) activated with CNBr.
  • the column was equilibrated with TMB buffer (10 mM Tris-HCl, pH 7.0, 2 mM MgC1 2 , and 0.5 mM DTT), and a solution of a purified polypeptide diluted 10 times in the same buffer was put on the column to allow binding of the polypeptide to the column.
  • the column was washed throughly with the same buffer, and after substances that had bound nonspecifically were removed in this way, elution was done with slowly increasing concentrations of potassium chloride. A portion of each fraction was put on an SDS-polyacrylamide gel and electrophoresed, and the polypeptide in question was found in the fractions that had been eluted with potassium chloride at the concentrations of 40 to 60 mM.
  • phosphorylated myosin final concentration, 0.2 mg/ml
  • actin final concentration, 0.1 mg/ml
  • tropomyosin final concentration, 30 ⁇ g/ml
  • phosphorylated myosin final concentration, 0.2 mg/ml
  • actin final concentration, 0.1 mg/ml
  • tropomyosin final concentration, 30 ⁇ g/ml
  • a buffer that contained 20 mM imidazole-HCl (pH 7.2), 0.1 mM DTT, 100 mM KCl, 2 mM MgC1 2 , 1 mM ATP, and 0.1 mM CaC1 2 .
  • the purified polypeptide was added and the mixture was kept at 30°C for 5 minutes.
  • trichloroacetic acid was added to the final concentration of 10% to stop the reaction.
  • the reaction mixture was centrifuged, and the amount of free phosphate in the supernatant was assayed by the method of Youngburg (BBRC, 132 , 645-651, 1985).
  • the inhibition caused by the polypeptide was found from the difference in the amount of free phosphate related by ATPase in the presence and absence of the polypeptide. Both of these activities were found in type I, type II, 312AA, 122AA, 118AA, and 116AA.
  • the functional unit of human caldesmon polypeptide was found to be 116AA.
  • this invention provides the complete amino acid sequence of human caldesmon, and the complete DNA sequence that codes for human caldesmon, and it identifies the functional unit of caldesmon activity. These genes and the polypeptides are useful in the fields of biochemistry, medicine, including diagnosis, and the like.
  • Figure 1 is a restriction map of the cDNA that codes for type I and type II caldesmon (CaD).
  • Figure 2 is a figure that shows the relationships of type I, type II, 312AA, 118AA, and 90AA.

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Claims (6)

  1. Isoliertes und nicht-natürlich vorkommendes Polypeptid mit Calmodulin-Bindungsaktivität und Actin-Bindungsaktivität, welches die in SEQ ID Nr. 1 gezeigte Aminosäuresequenz in einem Polypeptidmolekül umfaßt.
  2. Isoliertes und nicht-natürlich vorkommendes Polypeptid nach Anspruch 1, welches die in SEQ ID Nr. 1, SEQ ID Nr. 2, SEQ ID Nr. 3 oder SEQ ID Nr. 4 gezeigte Aminosäuresequenz aufweist.
  3. Isoliertes Gen, weiches für ein Polypeptid nach Anspruch 1 oder 2 kodiert.
  4. Isoliertes Gen nach Anspruch 3, welches die in SEQ ID Nr. 7 gezeigte DNA-Sequenz in einem Genmolekül umfaßt.
  5. Isoliertes Gen nach Anspruch 4, welches die in SEQ ID Nr. 7, SEQ ID Nr. 8, SEQ ID Nr. 9 oder SEQ ID Nr. 10 gezeigte DNA-Sequenz aufweist.
  6. Isoliertes Gen nach Anspruch 3, welches das Gen von Anspruch 4 oder 5 unter stringenten Bedingungen hybridisieren kann.
EP19920302591 1991-03-29 1992-03-25 Calmodulin bindendes Protein Expired - Lifetime EP0506377B1 (de)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP8910691 1991-03-29
JP89106/91 1991-03-29
JP3358040A JP2919144B2 (ja) 1991-03-29 1991-12-27 ポリペプチド
JP358040/91 1991-12-27

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EP0506377A2 EP0506377A2 (de) 1992-09-30
EP0506377A3 EP0506377A3 (en) 1993-04-14
EP0506377B1 true EP0506377B1 (de) 1997-12-10

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Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH07135980A (ja) * 1993-11-19 1995-05-30 Chisso Corp シュードモナス菌リパーゼ遺伝子
US5624902A (en) * 1995-06-07 1997-04-29 Torrey Pines Institute For Molecular Studies Peptide inhibitors of calmodulin
US20050163755A1 (en) * 2003-12-15 2005-07-28 Moy Alan B. Methods and compositions related to 1-caldesmon
WO2006005585A2 (en) * 2004-07-12 2006-01-19 Geneprot, Inc. Secreted polypeptide species differentially expressed during pregnancy

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EP0506377A3 (en) 1993-04-14
DE69223434D1 (de) 1998-01-22
EP0506377A2 (de) 1992-09-30

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